The DNA base excision repair (BER) process, active in both nuclei and mitochondria, is primarily responsible for removing single-strand breaks and oxidized, alkylated and abnormal base lesions from their genomes. Such lesions are induced by environmental agents, particularly reactive oxygen species (ROS), which are continuously generated in the mitochondria. The mitochondrial genome is more susceptible to ROS than the nuclear genome. AP-endonuclease (APE) plays a key role in BER by removing both abasic (AP) sites, and 3' blocking groups at DNA single- strand breaks. Both of these lesions are generated either directly by ROS or indirectly during repair of oxidized bases. Two types of APE, Xth and Nfo, first discovered in E.coli, are present in both bacteria and yeasts, while two APEs, both of the Xth type, have been identified in mammals. The major and better characterized human APE, hAPE1, is a multifunctional protein with additional transcriptional regulatory functions. Our preliminary studies indicate that the recently cloned hAPE2, whose activity in vitro or in vivo has not yet been characterized, is localized in the mitochondria. Deletion of apn2, the APE2 ortholog in the fission yeast S. pombe, increases its sensitivity to low levels of ROS and alkylating agents. However, homologous recombination, active in mitochondria of yeast but not of mammals, and yeast's ability to produce energy by fermentation, may make the mitochondrial BER process less critical in yeast than in mammals. The central focus of this continuing project is a comprehensive characterization of nuclear and mitochondrial APEs in mammalian cells, including their structure-function relationships, in vivo activities, and roles in BER. The specific aims of this project for the nest funding period are to: 1) characterize hAPE2 and S. mombe apn2, elucidate their structure- function relationships, and test in the in vivo role of apn2 in mitochondrial protection in the absence of recombination; 2)generate conditional knockout mutations of the mouse APE2 gene, first in embryonic stem cells, and then in transgenic mice, in order to examine its in vivo role; and 3) characterize the sites of phosphorylation in hAPE1 and the effects of phosphorylation on its structure and enzymatic activities. The long-range goal of this project is to understand the mechanism of oxidative damage repair via the BER pathway for both nuclear and mitochondria genomes, and to determine how modulation such repair may prevent carcinogenesis, other pathologies, and aging.